title: Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.
abstract: We previously demonstrated the presence of a soluble form of full-length Alzheimer's amyloid precursor protein (APP) in the lumen of adrenal medullary chromaffin granules (CG). Furthermore, full-length APP is released from CG membranes in vitro at pH 9.0 by an enzymatic mechanism, sensitive to protease inhibitors [Vassilacopoulou et al. (1995) J. Neurochem. 64, 2140-2146]. In this study, we found that when intact CG were subjected to exogenous trypsin, a fraction of APP was not digested, consistent with an intragranular population of APP. To examine the substrate-product relationship between membrane and soluble full-length APP, we labeled CG transmembrane APP with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic probe, specific for membrane-spanning domains of proteins. APP released from the membranes at pH 9.0 was not labeled with [125I]TID. In addition, this APP was not biotinylated in intact CG. Combined, the results indicate that APP released from CG membranes derives from a unique nontransmembrane population of membrane-associated APP, located in the lumenal side of CG membranes. Dithiobis(succinimidylpropionate) (DSP) cross-linking indicated that APP in CG is situated in close proximity with other proteins, possibly with APP itself. APP complexes were also detected under nonreducing conditions, without DSP cross-linking. These results, combined with our previous studies, indicate that full-length APP within CG exists as three different populations: (I) transmembrane, (II) membrane-associated/nontransmembrane, and (III) soluble. The existence of nontransmembrane populations suggests that putative gamma-secretase cleavage sites of APP, assumed to be buried within the lipid bilayer, could be accessible to proteolysis in a soluble intravesicular environment.
authors.affiliation: Department of Psychiatry, Mount Sinai School of Medicine, New York 10029, USA.
authors.forename: N
authors.initials: N
authors.lastname: Tezapsidis
authors.forename: H C
authors.initials: HC
authors.lastname: Li
authors.forename: J A
authors.initials: JA
authors.lastname: Ripellino
authors.forename: S
authors.initials: S
authors.lastname: Efthimiopoulos
authors.forename: D
authors.initials: D
authors.lastname: Vassilacopoulou
authors.forename: K
authors.initials: K
authors.lastname: Sambamurti
authors.forename: T
authors.initials: T
authors.lastname: Toneff
authors.forename: S
authors.initials: S
authors.lastname: Yasothornsrikul
authors.forename: V Y
authors.initials: VY
authors.lastname: Hook
authors.forename: N K
authors.initials: NK
authors.lastname: Robakis
mesh_terms: D000313:Adrenal Medulla; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001389:Azirines / Q000378:metabolism; D019921:Biotinylation; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002837:Chromaffin Granules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003432:Cross-Linking Reagents; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels; D014357:Trypsin / Q000494:pharmacology
chemical_list: D016564:Amyloid beta-Protein Precursor; D001389:Azirines; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D019920:Photoaffinity Labels; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D014357:Trypsin